Pyrogens - Prof. J.

Vijaya Ratna Jayanti

For the parenteral products, the most usual vehicle is water. Water is most abundantly
available, cheap, safe, non-toxic and non-irritating. But anything that is to be
administered parenterally has to be sterile and apyrogenic. Apyrogenesity means lack
of pyrogens. Pyrogens are the metabolic products of bacteria. In making a vehicle
suitable for parenteral preparation, we may sterilize it by autoclaving, which would kill
the vegetative organisms as well as the spores. But the water once had organisms
living in it and their metabolic products now stay in the water. These products are not
destroyed by autoclaving. Since they cannot be destroyed at the usual autoclaving
temperature, they must be prevented from getting into water. So the water being
taken for parenteral preparation must be apyrogenic.
Pyrogens can cause a lot of damage, if they are injected into a human being. Hence
every batch of injections is tested for the presence of pyrogens. The word "pyrogens"
means, "fever producing".

What is the Definition of Pyrogen?

Pyrogens are fever producing substances, which are metabolic products of
microorganisms. Chemically, they are lipid substances associated with a carrier
molecule, which is usually a polysaccharide. The carrier may also be a peptide. These
carriers increase the solubility of the lipid. Pyrogens are produced by many
microorganisms including bacteria, yeasts and moulds. Most potent pyrogens are the
endotoxins produced from the cell walls of the Gram- negative bacteria. Pyrogens have
a high molecular weight, often, more than 1,000,000. 1
What is the source of Pyrogen?
Sources of Pyrogens: Solvents, drugs, additives apparatus used in manufacture,
containers; all or any of these may be sources of pyrogens. The method of storage in
between preparation and sterilization also may cause the development of pyrogens.
Hence every item must be apyrogenic and method of storage must not allow any
bacterial growth.
What PYROGENS do?
If unintentionally, pyrogens are injected into a patient, they may bring about the
following physiological changes:
Erythema at the injection site
Pain in the legs and trunk
General discomfort
High temperature

Pyrogens, if present in large volume parenterals, can be especially dangerous, as they

would be present in large quantities and are given intravenously. Pyrogens in large
volume parenterals may make the patients very ill and the effect of rise of
temperature may be fatal. Hence there is a need for testing parenteral solutions for
their apyrogenesity and Pharmacopeias make it compulsory.
What is the history of Pyrogens?
In the years gone by, there was only one way in which pyrogens were tested for, i.e.,
by the Rabbits' test. But today much easier methods such as the limulus amoebocyte
lysate test are available for testing for pyrogens. If we look at the history of pyrogens,
we find that since the time of introduction of intravenous therapy, in 1656, by Sir
Christopher Wren, the awareness of a fever producing agent was there. The
documented research into the development of medicines contains many references to
episodes of fever following intravenous infusions. But the scientists could not find the
mechanism by which intravenous therapy was producing fever. Since fever frequently
followed sepsis in wounds, physicians thought that the tissues in the wounds were
decomposing, then putrefying by fermentation and in the process were causing the
fever. Louis Pasteur (1822-1895), discovered that bacteria caused fermentation. Then
scientists associated fever and pyrogens with fermentation and with bacteria. But they
had no idea about the origin of pyrogens, whether they were produced by bacteria or
whether they were a part of bacteria.
Research into the nature of pyrogens started showing good results in 1894, when
Centanni extracted pyrogens from many types of bacteria. He extracted pyrogens from
Esherichia coli too. Scientists started looking into the chemical and physiological
nature of pyrogens. Centanni showed that pyrogens were not proteinous and that they
were not susceptible to heat. It was the publications of Hort and Penfold, in the year
1912, that provided the best understanding of the nature of pyrogens. They started
the Rabbit Assay and standardized it for fever. By correlating the fever effect in rabbits
with the extent of pyrogens, and by characterizing the bacteria by staining as Gram
positive and Gram negative Hort and Penfold could prove that the pyrogenic bacteria
were predominantly Gram negative whereas the Gram positive were mostly
apyrogenic. Then they could correlate between the bacterial count in the injected
distilled water and the extent of fever caused and also prove that dead bacteria also
caused fever. Then, Hort and Penfold, concluded that the real cause of injection fevers
was a filterable substance which was stable to heat, and was produced by bacteria.
Seibert's investigations in the years1923-1925, proved these findings conclusively.
Seibert could also develop a process for the production of apyrogenic infusion fluids.
Hospital pharmacists were very happy to follow the method introduced by Siebert to
produce large volume parenterals that were very safe for use in patients. Seibert's
methods also launched the large commercial LVP solutions industry. Today's huge LVP

production industry depends on the availability of large volumes of sterile and

apyrogenic water which is produced by distillation.
Sponsored Links
A typical production procedure for manufacture of injectables goes like this:
Apyrogenic raw materials are dissolved in apyrogenic water for injection. Mixing is
done in depyrogenated tanks. The prepared solution is dispensed into depyrogenated
containers. These containers are immediately autoclaved in order to prevent the
growth of any microorganisms that might be present in the solution.
What is the Nature of Pyrogens?
A pyrogen is a fever producing substance. The most important pyrogen is the
endotoxin produced by the Gram negative bacteria. Some pyrogens are there, which
are not of microbial origin. They are in fact pharmacological agents, such as bleomycin
and colchicines. But it is the microbial pyrogens that pose a problem to the
manufacturers. While many organisms including live and dead bacteria, fungi, viruses,
malarial parasites, and the bacterial products such as streptococcal exotoxins,
staphylococcal enterotoxin, bacterial endotoxin lipopolysaccharide, and fungal
products can act as pyrogens, it is the Gram negative bacteria produced endotoxin
that is the most significant pyrogen for the manufacturers of injectables. This
endotoxin is the most significant because its lipopolysaccharide component is very
toxic in very small quantities. A quantity as small as 1ng/kg of the USP Reference
endotoxin can cause fever.
Fever inducing mechanism:
Endotoxin pyrogen enters the bloodstream.
It binds to Lipopolysaccharide (LPS) binding protein (LPB). LPB takes it to the
reticuloendothelial system (RES). The receptor cells in the RES are the circulating
mononuclear
cells.
This attachment to the receptor cells causes the production of proinflammatory
cytokines.
These cytokines are interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factora
(TNFa).
These factors produce inflammation and fever.
Nature of Bacterial Endotoxin: This endotoxin is present almost everywhere and is very
difficult to remove. Its nature and omnipresence is as following:
This is a high molecular weight complex molecule. This comes from the outer cell wall
membrane of gram negative bacteria. These GNB keep shedding the endotoxin into
the
environment
in
which
live.
The endotoxin is shed by the bacteria as they live, as they multiply and also after they
die and disintegrate.
So endotoxin may be present in a system in a form associated with live organisms or
unassociated, in a free manner. Endotoxin associated with microorganisms may be

removed by microporous sterilizing filters. But unassociated endotoxin passes through

these filters also. It cannot be destroyed by autoclaving as it is heat stable. Endotoxin
can be found in food materials, in ground waters, in soil and in substances. It is found
on
laboratory
glassware,
research
apparatus,
and
water
baths.
It can be found in all places where moisture may be there.
Chemical Nature of endotoxin: Bacterial endotoxins contain lipid, protein and
carbohydrate.
The primary chemical structure is a polysaccharide structure which is covalently bound
to a lipid, called lipid A. Because of this structure, the bacterial endotoxin is called as
lipopolysccharide
or
LPS.
The lipid A part is in the bacterial cell wall. The polysaccharide projects into the
environment.
The polysaccharide consists of two parts; a core oligosaccharide which is connected to
Lipid A and a longer oligosaccharide O- specific chain which is attached to the core.
This second oligosaccharide O- specific chain is the variable part of the molecular
structure. The minimum variable component is the lipid A. Lipid A consists of a
disaccharide of glucosamine. The glucosamine is highly substituted with ester linked
and amide linked long chain fatty acids. Each fatty acid has a backbone of 14 carbon
atoms. Lipid A is responsible for the harmful and useful activities of endotoxin.
References:
1. The Theory and Practice of industrial Pharmacy, Leon Lachman and Herbert A.
Lieberman, Special Indian Edition, 2009, CBS Publishers and distributors, page 641.
2. James Swarbrick, Encyclopedia of Pharmaceutical Technology, Volume 1,Third
edition, , Informa Health care, Newyork: 2007: pages 3052- 3053